The occurrence of crystals in tissue culture, particularlyin large numbers, is a rare finding, as reflected in thesparse literature on the subject. Recently Rose has shownthat embryonic chick tissue cultured in vitro under a complete sheet of cellophane membrane (‘dialysiscompartment') gives rise to a variety of extraordinary biologiccrystals. He also has stated that human embryonic andneoplastic tissues display a “capacity to give rise to similarforms― (9, 10). Subsequent studies by Rose (ii, 12)showed the same type of crystals in cultures of humanfetal thymus, adult gingiva, melanoma, and carcinoma ofthe thyroid. Concurrent with the latter report, an essentially identical crystalline deposit was found in our ownin vitro study of mammary carcinoma (C3H/HeJ mice).Both neoplastic and non-neoplastic tissues were associatedwith the occurrence of the crystals and, in contradistinctionto the work of Rose, a dialysis compartment was notrequired. Of additional interest is the failure of kidneyexplants to display these crystals.

Although the number of animals studied and the totalexplantation experience with these tissues is relativelysmall, the findings have been sufficiently uniform towarrant a preliminary report. The unique aspects of theobservations are enhanced by the failure to find similarcrystals in hundreds of primary cultures of various tissues,including many human neoplasms, such as breast and lungcarcinomas, despite the employment of identical technics.

MATERIALS AND METHODS

Female C3H/HCJ mice were used in this experiment.Animals were given dry food ad libitum (‘Success'Mouseand Rat Cubes with 3 % bentonite, Golden State MillingCompany, Downey, California). Two groups of animals

i This work was supported by the National Institutes of HealthGrant@ CA 06946-01 and by the California division of the American Cancer Society.

Received for publication September 2, 1964.

were used. An experimental group was maintained on amixture of 3 parts distilled water (Matillija Water Company, Ventura, California) and 1 part filtered sea water(suburban Pacific Ocean 100 yards off shore, Dexiglass no.225 filter, Dexter, Connecticut). The control group received distilled water throughout the experiment. Bothgroups were given water ad libitum. Animals were keptin steel cages at room temperature.

At intervals, animals with tumors and without detectable tumors were sacrificed. The tissues to be used forin vitro studies were dissected under aseptical conditionand 6-8 fragments of approximately 1—2mm cubed werecultured directly on glass in Rose chambers (7, 8) under a15-mm wide strip of dialysis cellophane membrane.Hence the nutrient medium was in direct continuity withthe explant environment. Cultures were also prepared ontube slips in Leighton tubes (5) without an anchoringsubstance such as collagen, plasma clot, or cellophanestrips. The same technic has been reported previously(6). Representative fragments of viable tumor were selected for explantation. In addition, 3 other tissues, lung,kidney, and spleen, were selected from both tumor andnontumor bearing animals (Table 1). These tissues werechosen because of several factors, but particularly for theirlarge size and ease of cultivation in vitro. Two culturemedia were used for each type of tissue : (a) Regular medium (RM) consisted of Eagle's (2) double strength aminoacid medium, 20 % calf serum and 5 % beef embryo ultrafiltrate EE-50 (Microbiological Associates Inc., Bethesda,Maryland). (b) Experimental medium (EM) differedfrom the RM only in the balanced salt solution component; regular Earl's balanced salt solution (3) was sub

stituted with 1 part sterile sea water and 2 parts steriletriple distilled water to which NaHCO3 was added (2.1gm/liter)2. Also incorporated in both media were 500mg/100 ml of glucose and streptomycin and penicillin,

2 After Lewis, M. R., Anat. Rec., 10:287, 1915—16.

214

The Occurrence of Biologic Crystals in Tumor andNontumor Cultures of C3H / HeJ Mice'

ARNIS RICHTERS AND RUSSELL P. SHERWIN

(University of Southern California School of Medicine, Department of Pathology, Los Angeles, California)

SUMMARY

Crystalline structures of various types have been found in primary tissue culturesof neoplastic and non-neoplastic tissues from C3H/HeJ mice. All of the cultures ofmammary carcinoma tissue contained the crystals. The crystals were found invarying incidence in the cultures of several other tissues with the exception of thosefrom the kidney. A ‘dialysis compartment' was not essential for the formation ofthe crystals. The development of the crystals was accelerated by a modification ofthe culture medium.

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Giom.TUMOR-BEARING

MICENONTUMOR-BEARINGMICENo.

oftumors

explantedCrystalsNo.

oflungs

explantedCr@rstalsseen inNo.

ofspleens

explantedCrystalsseen inNo.of

kidneysexplantedCr@rstalsseen inNo.

oflungs

explantedCrystalsseen inNo.of

spleensexplantedCrystalsseen inNo.

ofkidneys

plantedcx

CrystalsseeninExperimental

Exp.med.

Reg.med.

ControlExp.med.

Reg.med.5

5

5

55

5

5

54

4

3

30

0

1

02

2

2

22

2

2

12

2

2

20

0

0

02

2

2

21

0

2

12

2

2

22

1

1

12

2

2

20

0

0

0

RICHTERS AND SHERwIN—Crystals in Cultures of C3H/HeJ Mice 215

TABLE 1CRYSTAL OCCURRENCE IN ROSE CHAMBER CULTURES

each at 100 units/mi. Some Rose chambers withouttissues, but containing only the nutrient medium, werealso used. Media in culture chambers were changedwhenever the pH became quite basic or acidic as indicatedby phenol red indicator. On the average this occurredtwice a week.

RESULTS

Different crystalline structures were observed in severalcultures after 4 weeks in vitro. Six distinct crystallineforms were recognized : (a) rhomboids of varying sizes,(1?) tubular forms of different length and diameter, (c)ribbons of different length and width, (d) helices of different sizes, (e) filamentous forms, and (J) needle-like structures (Figs. 1—6). Hexagonal forms were seen infrequently(Fig. 7). Usually the crystalline structures found in bothcontrol and experimental groups appeared in the EM medium first, near the explanted fragments. In some casesthe crystalline structures were associated with a peculiarand n@tixedtype of deposition away from the fragment.After 5 to 6 weeks in vitro, crystals could be found throughout the chamber and were seen frequently on the surfaceof the cells (Figs. 8 and 9). In addition, some of thecultures showed crystalline structures which were apparently within the explant itself (Figs. 10 and ii). Frequently, the crystalline structures floated freely in themedium when the chamber was moved. In a few instances, numerous crystalline structures were observedwithin Leighton tubes. An attempt at crystal formationby acidification of the media in a cell free chamber wasnot successful. The occurrence of crystals is summarizedin Table 1.

Crystalline structures in tumor cultures.—All tumor cultures in vitro showed crystaffine structures after 3 to 6weeks in vitro. All of the crystalline forms described wereobserved. The crystals were first seen in the experimentalmedium; 4 to 6 days later, they were noted in the regularmedium. A preferential site of deposition for the differentcrystal types was not observed.

Spleen cultures.—All spleen cultures from tumor-bearingmice with one exception showed crystalline structures.In 3 cases, the crystals were first observed in spleen culturesand appeared in the other cultures of the same animal

4-6 days later. Again the crystals were first seen in thecultures in the experimental medium. The spleen culturesfrom nontumor bearing mice showed crystaffine structuresabout a week later than the cultures of spleen from thetumor-bearing mice. However, cultures from the spleenof one animal of the control group did not show any crystalformation.

Lung cultures.—At a time when all tumor cultures andall but one of the spleen cultures of the tumor-bearinganimals showed crystalline structures, only 1 lung culturewas positive for crystals. In the nontumor bearing group,4 lung tissue cultures had crystals.

Kidney cultures.—The kidney cultures did not showcrystalline forms in either group at any time during thestudy. Both Leighton tubes with tube slips and Rosechambers with cellophane strips were used.

DISCUSSION

The foregoing observations have demonstrated that thecrystalline structures appear not only in cultures of tumortissues but also in some cultures of apparently normal,mature tissues of the same animal. Although crystallineforms described by Rose are remarkably identical to thosefound in our study, the ‘dialysiscompartment' (nonperforated cellophane sheet) which was necessary for crystaldevelopment in his study, was not required in our investigation. Significantly, crystals were also observed in theLeighton tube preparations, but they were not studied indetail.

Additional findings of interest from our studies are:(a) Apparently significant differences in the time of crystaldevelopment were related to the use of media differingonly in the replacement of the balanced salt solution by asea water counterpart. (b) Crystals did not occur in anyof the tissue cultures of kidney despite a relatively uniformoutgrowth of epithelial cells. (c) The spleen cultures oftumor-bearing mice developed crystals 4—6days earlierthan thespleen culturesof nontumorbearing mice. (Therewas no exception to this finding.) (d) No significant differences were noted in the occurrence of crystals withother tissues although there was a strong suggestion thatthe lung cultures of tumor-bearing mice produced lesscrystals than the nontumor ones. In this preliminary

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Cancer Research216study, no attempt has been made to determine the natureand origin of the crystals or to quantitate them. We haveobserved a close association between the crystals and thetumor cells without any apparent damage to the cells(Figs. 8, 9, and 12).

The C3H/HCJ mouse strain is highly inbred and develops an almost 100 % incidence of mammary cancer.Although there have been many studies of mouse mammary cancer, both in vivo and in vitro (4, 13, 15), crystalformation has not been recorded in either type of study.A specific search for the crystals in the explanted fragments(the fragments from Leighton tubes were formalin fixedafter varying in vitro periods) is planned since similarcrystals have been described in tissues (in vivo) by Yang(16). With respect to Yang's findings, it is of interestthat his illustrations, especially Figures 6 and 8, are consistent with tubular and helical structures, although detailsas to these specific forms were not given. It is also pertinent to note that crystals of a similar type may form invivo and be discharged from the tissue on explantationin vitro, as implied in the report by Albores-Saavedra (1).

Both the nature of the crystals and the reasons for theiroccurrence are not clear at the present time. A strongprotein relationship has been mentioned by Rose (12) andrecently, Shelton has reported similar hexagonal-rhomboidal forms, as ‘cholesterol crystals' (14).

ACKNOWLEDGMENTS

We wish to express our appreciation to George MacGinitie,Emeritus Professor, California Institute of Technology, for hishelp and encouragement.

REFERENCES

1. ALBORES-SAAVEDRA,J.; RosE, G. G.; IBANEZ,M. L.; RUSSELL,W. 0.; GREY, C. E., AND DMOCHOWSKI,L. The Amyloid inSolid Carcinoma of the Thyroid Gland. Lab. Invest., 13:77—93,1964.

2. EAGLE, H. Nutritional Needs of Mammalian Cells in TissueCulture. Science, 122@501—04,1955.

3. EARLE, W. R. Production of Malignancy in Vitro. IV. TheMouse Fibroblast Cultures and Changes Seen in the LivingCells. J. Nati. Cancer Inst., 4:165—212,1943.

4. FOULDS,L. The Histological Analysis of Mammary Tumors ofMice. Ibid., 17:701—802,1956.

5. LEIGHTON, J. The Growth Patterns of Some TransplantableAnimal Tumors in Sponge Matrix Tissue Culture. Thid.,15:275—93,i954.

6. RICETERS,A., ANDSHERWIN,R. P. The Behavior of Lymphocytes in Primary Explants of Human Lung Cancer in Vitro.Lab. Invest., 13:1520—29,1964.

7. ROSE, G. G. A Separable and Multipurpose Tissue CultureChamber. Texas Repte. Biol. and Med., 12:1074—83, 1954.

8. ROSE,G. G.; POMERAT,C. M.; SHANDLER,T. 0.; ANDTRUNNELL, J. B. A Cellophane-strip Technique for CulturingTissue in Multipurpose Culture Chambers. J. Biophys. Biochem. Cytol., 5:761—64,1958.

9. ROSE, G. G. Helical Megamolecules : Products of Tissue Culture. Excerpta Med., 16: Sect. 1, 800, 1962.

10. . Biological Crystals and Particles Produced in TissueCulture. I. Introduction. Cancer Research, 23@79-.284, 1963.

11. . Phase-contrast Microscopy in Living Cells. J. Roy.Microscop.Soc., 83.'97—114,1964.

12. . Biological Crystals and Particles Produced in TissueCulture. II. Enzymatic Responses and Environmental Transformations. Cancer Research, 24:1159—93,1964.

13. SANFORD,K. K. ; DUNN, T. B. ; WESTFALL,B. B. ; COVALSKY,A. B. ; DUPREE, L. T. ; ANDEARLE, W. R. Sarcomatous Changeand Maintenance of Differentiation in Long-term Cultures ofMouse Mammary Carcinoma. J. Natl. Cancer Inst., 26:1139—83, 1961.

i4. SHELTON,E. ; T. T. OTANI; ANDFALES, H. M. Accumulationof Cholesterol Crystals in Diffusion Chambers Implanted inMice. Nature, 202:1229-30,1964.

15. STANTESSON,L. Characteristics of Epithelial Mouse TumorCells in Vitro and Tumor Structures in Vivo.A ComparativeStudy. Acta Pathol. and Microbiol. Scand. Suppi., 23-24:1—237,1935.

16. YANG,Y. H., ANDCAMPBELL,J. S. Crystalline Excrements inBronchitis and Cholecystitis of Mice. Am. J. Pathol., XLV:337—345,1964.

FIG. i. An aggregate of rhomboid crystals in mouse mammarytumor culture maintained on experimental medium (EM) . Tumorobtained from the experimental animal. Culture 34 days invitro. Phase-contrast photomicrograph. x 400.

FIG. 2. Rhomboid and tubular crystals in mouse mammarytumor culture maintained on EM medium. An experimentalgroup animal. Culture 34 days in vitro. Phase-contrast photomicrograph. X 160.

FIG. 3. Helical, ribbon, and tubular forms of crystals in mousemammary tumor culture from a control animal. Note the broad

ribbon in upper left corner and the smaller ribbon-like crystalscloser to the helix. Culture 34 days in vitro. Phase-contrastphotomicrograph. X 160.

FIG. 4. Helical crystal and some rounded cells in culture ofmouse mammary tumor from experimental animal. Culture 30days in vitro. Phase-contrast photomicrograph. X 400.

FIG. 6. Various crystalline forms observed in spleen culture oftumor-bearing animal. Note the tubules, ribbons, rhomboids,and filamentous forms. Culture 48 days in vitro. Phsse-contrast photomicrograph. X 100.

FIG. 6. Filamentous, needle-like, and fine tubular crystals inmouse mammary tumor culture from experimental animal. Culture 34 days in vitro. Phase-contrast photomicrograph. X 160.

Vol. 25, February 1965

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Fiu. 7. Hexagonal and rhomboid crystals in tumor culturefrom experimental animal. Culture 34 days in vitro. Phasecontrast photomicrograph. X 160.

FIGS. 8-9. Crystals in close association with mouse mammarytumor cells. The crystals are very transparent and the cellsunderneath the crystals can be seen. Culture 34 days old.Phase-contrast photomicrograph. X 160.

FIG. 10. Dense crystal aggregation around the explanted fragment of tumor. Some crystalline structures appear to be in thefragment. The periphery of tumor cell outgrowth can be seenin the lower left corner. Culture 34 days in vitro. Phase-contrast photomicrograph. X 100.

FIG. 11. Crystals surrounding and almost completely obscuringthe explanted tumor fragment. The disorderly arrangementand closely packed nature of the crystals is consistent with deposition within and around the fragment. Culture 34 days in vitro.Phase-contrast photomicrograph. X 100.

FIG. 12. An aggregate of rhomboid crystals in periphery ofmouse mammary tumor cell outgrowth. Culture 34 days in vilro.Phase-contrast photomicrograph. X 160.

218

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1965;25:214-219. Cancer Res   Arnis Richters and Russell P. Sherwin  Cultures of C3H/HeJ MiceThe Occurrence of Biologic Crystals in Tumor and Nontumor

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